Bacterial capsular and outer membrane saccharides are abundant cell surface antigens that are generally accessible by antibodies and targets for protective immune responses. The surface carbohydrates of un-encapsulated Gram-negative bacteria (GNB) are lipopolysaccharides (LPS). LPS comprise a lipid A membrane anchor that links a single core polysaccharide to a polymer of O-polysaccharide (OPS) containing a number of repeated saccharide monomer units, which form short, intermediate, long or very long O-chains. While the core polysaccharide is mostly conserved within individual bacterial species, the O-polysaccharide can be variable and is used to distinguish serotypes.
Antibodies specific for the OPS of several important GNB human pathogens have protected against infection with the homologous pathogen in preclinical animal models [1,2] and clinical trials [3]. There is marked interest in the use of OPS as the basis of vaccines. However, isolated OPS are poorly immunogenic. Chemical linkage to protein carriers has improved their immunogenicity, and a functional boost response can be achieved by repeated administration of OPS conjugates. Despite their promise as vaccine antigens, the natural variability in polymer size represents a practical challenge in their commercial use. Longer chain and higher molecular weight saccharide haptens tend to be more immunogenic, but some bacterial strains produce the longer chains in lesser amounts, creating difficulties in developing different conjugation synthesis strategies, producing a uniform product, and producing high glycoconjugate immunogenicity. Size fractionation can be used to obtain the desired saccharide size; however, addition of a sizing step can introduce extraneous cost to the production process. Furthermore, the desired polysaccharide size may constitute only a minor proportion of the total saccharide population. A need exists for safe and efficient production of more numerous and longer OPS at less cost, for use in producing conjugate vaccines.
Wzz proteins are chain length regulators, expressed in the bacterial periplasm that control the activity of the Wzy OPS polymerase. The modal number of OPS monomer repeats produced by GNB using the Wzy LPS synthesis system is controlled by Wzz proteins. The protein structure and specificity of wzz family members varies between bacterial species. Some bacteria encode several different wzz genes, and their expression can be subject to control by growth phase and environmental conditions.